This is a basic science proposal designed to use in vitro electrophysiological and optical techniques to further our understanding of an extremely unusual form of cannabinoid dependent signaling discovered in my lab. Specifically, we have observed that cannabinoid receptor agonists potentiate action potential independent release of GABA in the dentate gyrus through a cannabinoid type I (CB1) receptor independent mechanism. Although recent years have brought an enormous wealth of new information about the specific roles of endogenous cannabinoids in CNS physiology, the effect of cannabinoid receptor agonists that we have discovered appears to be unique in numerous respects. First, it does not depend on activation of CB1, CB2, or vanilloid type I receptors, and is still present in CB1-/- animals. Second, it clearly modulates action potential independent exocytotic events without altering action potential dependent events, and third, it ultimately results in the facilitation (rather than inhibition) of exocytosis. While our preliminary data represent significant progress towards characterizing a new role for cannabinoids in CNS physiology, many fundamental questions regarding this new phenomenon remain. Therefore this proposal requests funds to continue our investigation in this area through implementation of two Specific Aims. Aim 1 will test the hypothesis that cannabinoid mediated facilitation of action potential independent exocytosis depends on a specific ligand receptor interaction with an as yet unidentified GPCR. Specifically, we hypothesize that a G1s -> AC -> cAMP -> PKA signaling cascade is involved. Aim 2 will test the hypothesis that anandamide or one of its metabolites will be the most effective endogenously available ligand for facilitation of action potential independent release, and that the best agonists will work well within a physiologically relevant concentration range. Collectively, these Aims are designed to provide data that have the potential to clearly establish the significance of our findings for the cannabinoid field, and that will directly facilitate a longer term and larger scale investigation of the phenomenon. PUBLIC HEALTH RELEVANCE: Nearly all the known central effects of endogenous cannabinoids are thought to be mediated by activation of a single type of receptor, the CB1 cannabinoid receptor. We have recently discovered a highly novel form of cannabinoid mediated signaling that does not depend on CB1 receptor activation, and that has unique effects on CNS physiology. The current proposal is designed to address several fundamental questions about this new signaling system that we hope will ultimately establish the overall significance (and potential) of the finding.